Activation of Stra 8 Gene During the Differentiation of Spermatogonial Stem Cells

Abstract:

Retinoic acid plays an important role in maintaining the structure and function in male testis. Recent studies showed that there is a group of genes that can be specially activated by retinoic acid during the development of male reproductive gland. The gene Stra8 (Stimulated by Retinoic Acid) was one of the gene in this group. In mouse，Stra8 is restrictively expressed in male germ line cells，and its function is related to the development of sperm. In order to investigate the feature of Stra8gene expression，the 1.4kb (-1407～+7) promoter region of Stra8 gene was amplified from mouse genomic DNA. The DNA fragment was then cloned into a promoter less vector to form the construct that contained the 1.4kb promoter region，and the reporter gene of EGFP that was regulated by 1.4kb Stra8 promoter. To investigate the specificity of Stra8promoter，the vector pStra8-EGFP was transfected into undifferentiated mouse stem cells such as ES-129，bone marrow mesenchymal stem cell (mMSC) and spermatogonial stem cell (mSSC). The results showed that the expression of GFP was only observed in the mSSC cells，which indicated that Stra8 gene was specially regulated in testis tissue. As the gene marker，vector pStra8-EGFP was then transfected to undifferentiated mMSC cells. After being selected by G418 for 2 weeks，the mMSC cells were induced by retinoic acid. After 12 hours induction，some induced cells started to express GFP protein，which was observed under the fluorescence microscope. At the same time，several stem cell specificity biomarkers such as Oct4，and spermatogonial stem cell biomarkers such as CyclinA2and Stra8 were detected in the induced cells by RT-PCR method. These results showed that the mMSCs would differentiated to spermatognial stem cells after induced by Retinoic Acid.